issn: 2233-601x (print) issn: 2093-6516 (online) study on ... · materials made of bovine or...

Korean J Thorac Cardiovasc Surg 2011;44:197-207 □ Basic Research □

DOI:10.5090/kjtcs.2011.44.3.197ISSN: 2233-601X (Print) ISSN: 2093-6516 (Online)

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*Department of Thoracic and Cardiovascular Surgery, Seoul National University Hospital, Seoul National University College of Medicine**Seoul National University Hospital Clinical Research Institute, Xenotransplantation Research Center

†This study was supported by a grant of the Korea Health 21 Research and Development Project, Ministry for Health, Welfare, and Family

Affairs, Republic of Korea (Project No.: A040004-006).Received: October 23, 2009, Revised: March 18, 2011, Accepted: May 10, 2011

Corresponding author: Yong Jin Kim, Department of Thoracic and Cardiovascular Surgery, Seoul National University Hospital, Seoul National

University College of Medicine, 28, Yeongeon-dong, Jongno-gu, Seoul 110-744, Korea(Tel) 82-2-2072-3638, (Fax) 82-2-745-5209 (E-mail) [email protected]

C The Korean Society for Thoracic and Cardiovascular Surgery. 2011. All right reserved.CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creative-

commons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Study on Genipin: A New Alternative Natural Crosslinking Agent for Fixing Heterograft Tissue

Jae Suk Yoo, M.D.*, Yong Jin Kim, M.D.*, Soo Hwan Kim**, Seung Hwa Choi**

Background: In cardiac surgery, especially in the reconstruction of vascular structures and intracardiac defects, glu-taraldehyde has usually been used as the reagent for fixing porcine or bovine pericardial tissues. But the well-known problem of calcification or cytotoxicity of glutaraldehyde motivates the search for a replacement. The aim of this study is to investigate the physical, mechanical, and biochemical characteristics of bovine pericardial tis-sues fixed with genipin, which is known to be a less toxic and more natural fixing reagent. Materials and Methods: Bovine pericardial tissues were fixed with different concentrations and conditions of glutaraldehyde and genipin. To determine the physical, mechanical, and biochemical differences among different concentrations and con-ditions, we divided the tissue into 18 groups by concentration, the addition of organic solvents, and the timing of adding the organic solvents, and compared the characteristics of each group. Results: Tensile strength, physical activity, and thermal stability tests revealed that the tissues fixed with glutaraldehyde were better with regard to mechanical strength and biochemical durability. However, the difference was not significant statistically. Conclusion: Genipin can be used as an alternative crosslinking agent for pericardial tissue, considering given its physical, me-chanical, biochemical characteristics and low cytotoxicity comparable to glutaraldehyde. However, further studies are needed on the immune reaction and the long term changes in genipin-fixed tissues in the human body.

Key words: 1. Xenograft2. Glutaraldehyde3. Bioprosthesis4. Pericardial patch

INTRODUCTION

In the field of cardiovascular surgery, there has been an ef-

fort to develop adequate prosthetic materials for car-

diovascular reconstruction, such as vascular conduits, valve

leaflets or patches for tissue defects. Until now, the biological

materials made of bovine or porcine pericardium have been

most widely used. These heterograft tissues must be bio-

logically stable and safe in the human body, so they should

be chemically modified or fixed to reduce immune or in-

flammatory reactions and to increase mechanical endurance

and tolerance [1]. The ideal condition of heterografts for car-

diovascular reconstruction is to be mechanically strong, toler-

ant of degeneration or calcification, easily manipulated by the

Jae Suk Yoo, et al

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surgeon’s hand, and also less cytotoxic and immunological [2].

Although several chemicals, including formaldehyde, gluta-

raldehyde, and epoxy compounds, that can fix biological tis-

sue, glutaraldehyde has usually been used. Because many

studies have been performed to evaluate the function, stabil-

ity, and safety of glutaraldehyde as a fixing crosslinking

agent, and to observe its long term use, there is sufficient

clinical knowledge and experience on glutaraldehyde. However,

glutaraldehyde still exhibits the drawbacks of high cytotox-

icity and calicification, and we also lack knowledge on heter-

otopic immune response to it in the human body. Therefore,

development of a new alternative fixation agent is required

[3]. For that reason, there have been attempts to use genipin,

a natural crosslinking reagent, as a fixation material [4].

Genipin is an agent extracted from gardenia fruit (Gardenia

jasminoides ELLIS). In traditional Chinese medicine, genipin

has been used as a treatment for DM or jaundice patients.

Many studies have reported on genepin’s much lower level of

cytotoxicity compared to glutaraldehyde [5].

The aim of this study was to evaluate whether genipin

could be used as an alternative crosslinking agent to gluta-

raldehyde for heterograft tissues. Thus, we compared the

physical, biochemical, and histological characteristics of heter-

ograft tissues (bovine pericardial tissues) fixed with genipin

and those fixed with glutaraldehyde. As is generally known,

the fixation of heterograft pericardial tissues with organic sol-

vent causes permanent changes because it suppresses the nu-

cleation of calcium phosphate and decreases the size and

amount of collagen, which is assumed to enhance the me-

chanical durability of heterograft pericardial tissues [6].

Therefore, we divided tissues into groups by concentration

of genipin and the addition of organic solvent and observed

changes in each tissue’s characteristics to prepare clinically

useful standards.

MATERIALS AND METHODS

1) Production of bovine pericardial patch

Bovine pericardium removed from cattle in an abattoir was

put into cold phosphate buffered solution (PBS, 0.1 M,

pH7.4), packed into a cooler and transported to the

laboratory. Every bovine pericardium was sterilized in 2% be-

tadine solution for 1 hour before being washed and stored in

normal saline.

2) Fixation

To observe differences in the characteristics of a pericardial

patch fixed with genipin and one with glutaraldehyde, parts

of a pericardial patch were treated with glutaraldehyde only

and, separately, glutaraldehyde with organic solvent added.

To identify differences according to the concentration of

genipin (0.1, 0.2, 0.3, 0.4, 0.6, and 1.0%) and other con-

ditions, we divided pericardial patchs into 16 groups by the

concentration of genipin, the addition of organic solvent, and

the timing of adding organic solvent. After fixation, the peri-

cardial patches were washed by PBS solution several times in

room temperature and stored in 4oC PBS solution. In the end,

we had prepared 20 pericardial patches for each group and

performed various tests with each patch.

All the fixation processes were performed at room

temperature.

(1) Fixation with glutaraldehyde (2 groups)Glutaraldehyde: 0.5% glutaraldehyde solution (PBS buffer,

pH 7.4) for 5 days → 0.25% glutaraldehyde solution for 1

week

Glutaraldehyde+Solvent: 0.5% glutaraldehyde solution for 5

days → 1% glutaraldehyde with organic solvent (75%

Ethanol+5% Octanol) solution for 2 days → 0.25% gluta-

raldehyde solution for 1 week

(2) Fixation with genipin (6 groups)0.1% Genipin: 0.1% genipin solution (PBS buffer, pH 7.4)

for 4 days

0.2% Genipin: 0.2% genipin solution for 4 days





(3) Fixation with genipin and organic solvent (10 groups)0.1% Genipin+Solvent: 0.1% genipin solution (PBS buffer,

pH 7.4) for 2 days → 0.1% genipin with organic solvent

(75% Ethanol+5% Octanol) solution for 2 days

0.2% Genipin+Solvent: 0.2% genipin solution for 2 days

→ 0.2% genipin with organic solvent for 2 days



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→ 0.3% genipin with organic solvent solution for 2 days







Solvent+0.3% Genipin: 0.3% genipin with organic solvent

(75% Ethanol+5% Octanol) solution for 2 days → 0.3% gen-

ipin solution for 2 days










3) Physical test

By palpation, extension, and suture, a single thoracic sur-

geon subjectively evaluated the characteristics of pericardial

patches fixed using each method.

Palpation was done by touching the pericardial patches

manually and measuring the softness of the pericardial

patches. Fresh bovine pericardium was scored 0 for standard,

1 for soft, 2 for slightly soft, 3 for moderately soft, 4 for

slightly stiff, and 5 for stiff.

Characteristics of and gaps in patches during suture were

described with a score indicating resistance against needling

the pericardial patch by a 5-0 Prolene.

The extensibility and gap was also estimated and scored by

extending the pericardial patch by pulling the suture needle

which had punctured the patch.

Using the sum of those scores, we compared patches fixed

with genipin to those with glutaraldehyde, patches fixed with

low and high concentrations of genipin, patches fixed with

and without organic solvent, and patches to which organic

solvent was added first to patches to which genipin was add-

ed first.

4) Tensile strength test

Using tensile strength test equipment and settings devel-

oped by our laboratory, we estimated and compared the ten-

sile strength of pericardial patches fixed in various ways [7].

The pericardial patches of each group were made into 5×50

mm patches and the tensile strength of 5-mm-wide patches

was estimated. The average of the estimated tensile strength

values was set as the representative value of the group. We

used a Digital Force Gauge (DS2-50N, Imada, Japan) for es-

timation of the tensile strength. The load speed was 100 mm/

min and measurement unit was N/5 mm2, MPa (megapascals).

5) Thermal stability test

The pericardial patches fixed in various ways were cut to

8×30 mm sections. While applying a continuous tensile

strength of 95 g, the patches were put into 55oC normal

saline. Until the point that the patches rapidly contracted, in

other words, the point of degeneration of collagen in the tis-

sues, the temperature of normal saline was increased with a

velocity of 1∼2.5oC/min. Then we measured and compared

the temperature of each group when the patched underwent a

sudden contraction.

6) Amino acid analysis

Pericardial patches of each group was cut into 10×10 mm

sections and put into a heat dryer (80oC, 650 G Fisher

Scientific, USA) for 24 hours after washing with normal

saline. We then removed the water in the pericardial patches

and measured the mass. Then those pericardial patches were

moved to 2 mL Effendorf tubes. 5 M HCL 1.5 mL was add-

ed to the tube and dried in the dryer for 24 hours. The color

of the HCl solution changed to yellow due to dissolution of

the specimen. We moved that yellow solution to the

Effendorf tube and dried it completely at room temperature.

The pellet obtained through the above-mentioned process was

sent to the Department of Laboratory Medicine for analysis.

They quantified 18 types of amino acids (aspartic acid (Asp),

glutamic acid (Glu), histidine (His), proline (Pro), threonine

(Thr), arginine (Arg), taurine (Tau), tyrosine (Tyr), methio-

nine (Met), valine (Val), phenylalanine (Phe), isoleucine (Ile),

leucine (Leu), and lysine (Lys)) by liquid chromatog-

Jae Suk Yoo, et al

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Table 1. Physical characteristics of pericardium determined by a single surgeon’s hand

Group Palpation Suture Extensibility Gap Score

Fresh pericardium

GA

GA

GA+Solvent

Genipin

0.1%

0.2%

0.3%

0.4%

0.6%

1.0%

Genipin → Selvent

0.1%

0.2%

0.3%

0.4%

0.6%

1.0%

Solvent → Genipin

0.3%

0.4%

0.6%

1.0%

2

3

4

2

3

2

2

3

2

3

3

3

3

4

4

4

4

4

4

2

3

3

4

5

3

2

2

2

2

4

3

5

5

4

4

5

4

4

3

4

4

3

4

2

2

3

3

3

4

4

4

4

4

4

4

5

5

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

7

10

11

9

12

7

6

8

7

8

11

10

12

13

12

12

13

13

13

GA=Glutaraldehyde.

Table 2. Tensile strength of pericardium (n=20)

Group

Tensile

strength

(MPa)

Tensile

strain at

break avg

(%)

Average

(n=20)p-value

Glutaraldehyde

Genipin

0.1%

0.2%

0.3%

0.4%

0.6%

1.0%

Genipin → Solvent

0.1%

0.2%

0.3%

0.4%

0.6%

1.0%

Solvent → Genipin

0.3%

0.4%

0.6%

1.0%

16.09±6.27

12.43±4.16

12.44±4.80

12.39±2.40

12.66±4.03

16.10±4.05

13.51±4.30

14.00±6.02

11.48±1.57

13.35±3.67

14.35±4.07

11.84±4.07

22.31±5.83

17.08±5.82

17.36±4.15

17.42±4.66

13.70±3.32

24.53±5.38

35.42±6.04

36.53±11.75

35.83±7.80

32.50±6.98

30.50±4.99

28.67±7.75

37.08±6.09

40.69±8.80

32.50±4.74

44.58±6.02

33.33±5.67

28.75±5.46

32.58±6.41

36.75±7.10

31.83±7.25

41.33±7.87

16.09±6.27

14.52±4.21

15.68±4.31

p=0.274

raphy/mass spectrometry (LC/MS, Waters, USA). To de-

termine the difference among crosslink characteristics con-

tingent upon concentration of genipin and organic solvent, we

separated the pericardial patches fixed with genipin only (0.1,

0.2, 0.3, 0.4, 0.6, and 1.0%), patches fixed with genipin after

being treated with organic solvent, and patches fixed with

genipin before being treated with organic solvent, and ana-

lyzed the residual amino acid in the pellet.

7) Statistical analysis

In the tensile strength test, the unit of tensile strength at

break was MPa and the unit of tensile strain at break was

‘mean value±standard deviation’ (%). In the thermal stability

test, the temperature at contraction of the pericardial patch

was indicated by oC, and each value was also indicated as

‘mean value±standard deviation’. The statistical difference

among the groups was verified by one-way analysis of var-

iance (ANOVA). The results wth a p-value＜0.05 were con-

sidered statistically significant.

RESULTS

1) Physical test

There was no significant difference in mechanical charac-

teristics between the pericardial patches fixed with genipin

and those with glutaraldehyde. Also, there was no clear rela-

tionship with the concentration of genipin. The pericardial

patches with organic solvent showed a greater tendency to be

durable and stiff (Table 1).

2) Tensile strength test

From the result of the tensile strength test, it seemed that

pericardial patches fixed with genipin or glutaraldehyde had

more tensile strength compared to fresh bovine pericardium.

However, there was no significant difference among the

groups fixed with glutaraldehyde, fixed with genipin only,

and fixed with genipin and treated with organic solvent

(p=0.274) (Table 2).


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Table 3. Thermal stability test (n=20)

GroupTemperature at

shrinkage (oC)

Average p-value

Fresh tissue

GA

GA+Solvent

Genipin

0.1%

0.2%

0.3%

0.4%

0.6%

1.0%

Genipin → Solvent

0.1%

0.2%

0.3%

0.4%

0.6%

1.0%

Solvent → Genipin

0.3%

0.4%

0.6%

1.0%

69.42±1.48

86.25±0

86.25±0

79.25±1.12

81.75±1.12

80.75±1.12

83.25±1.12

81.75±1.12

80.75±1.12

79.75±1.37

80.75±1.12

81.25±0

81.25±0

81.75±1.12

80.25±1.37

81.25±0

81.25±0

81.25±0

80.25±1.37

86.25±0

82.25±1.12

81.75±1.12p=0.183

GA=Glutaraldehyde.

3) Thermal stability test

The temperature at which the pericardial patch fixed with

genipin started to contract was 82.25oC, higher than the tem-

perature at which the fresh bovine pericardial patch starts to

contract (69.42oC), but slightly lower than the temperature for

a pericardial patch fixed with glutaraldehyde (86.25oC).

However, there was no significant difference among the

groups fixed with glutaraldehyde, fixed with genipin only,

and treated with organic solvent after being fixed with geni-

pin (p=0.183) (Table 3).

4) Amino acid analysis

The result of amino acid analysis showed a tendency to

decrease in amount of residual amino acid when the concen-

tration of genipin increased from 0.1% to 1.0%. Following

the increase in the concentration of genipin from 0.1% to

0.6%, the amount of Glu, Asp, His, Tyr, Met, Val, Gly, Arg,

Phe, Ile, Leu, and Lys showed a tendency to constantly

decrease. However, there was no significant difference be-

tween the 0.6% genipin and 1.0% genipin groups. It seemed

that more crosslinks were made with an increase in the con-

centration of genipin until the concentration of genipin

reached 0.6%, but there was no significant difference under

high concentrations of genipin, that is, over 0.6% (0.6%,

1.0%). Residual Ala was lowest in 0.4% and 0.6% genipin.

Ser showed many crosslinks in over 1% genipin.

In groups fixed with genipin after treatment with organic

solvent, there was no remarkable difference in the quantity of

residual amino acids according to the concentration of

genipin. Only the group fixed with the highest concentration

of genipin (1%) after treatement with organic solvent showed

a decrease in the amount of residual amino acids (Fig. 1).

Seeing that more crosslinking was observed in the group

without organic solvent, the crosslink between genipin and

amino acids seemed to be hydrophilic. As reported in Fig. 1,

groups of pericardial patches fixed with 0.3∼0.4% genipin

showed the least residual amount of most of the amino acids,

so it seems that crosslinking is most actively performed in

that range of concentration of genipin.

DISCUSSION

Glutaraldehyde is an agent for fixation to suppress the im-

munogenicity of heterograft tissue and enhance its durability,

and is being widely used as an inducer of biochemical cross-

linking in tissue [8]. So far, there have been many studies on

glutaraldehyde itself, and the biochemical structure and char-

acteristics of crosslinking induced by glutaraldehyde. Because

glutaraldehyde is generally used for the fixation of heterograft

tissue in the clinical field, clinical outcomes of glutaraldehyde

usage are also widely known. However, the cytotoxicity and

calcification of glutaraldehyde spurred the search for another

fixation agent to replace it. It has been thought that the calci-

fication of glutaraldehyde is caused by a reaction between

calcium in the human body and the aldehyde group in gluta-

raldehyde, which together combine with pericardial tissues.

Therefore, there have been various studies on methods for the

reduction of calcification, for instance, combining aldehyde

groups with other agents in advance and blocking the re-

action with calcium [9]. Furthermore, because of the cytotox-

Jae Suk Yoo, et al

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Fig. 1. Residue patterns of each amino acid according to each fixation method.


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Fig. 1. Continued.

Jae Suk Yoo, et al

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Fig. 1. Continued.

Fig. 2. Structure of genipin.

icity of glutaraldehyde itself, studies on genipin, which can

generate crosslinks naturally and may replace glutaraldehyde,

are now being performed.

Genipin, which has a C11H14O5 molecular structure (Fig. 2), is

iridoid glycoside, which is one of the main ingredients in garden-

ia fruit (Gardenia jasminoides ELLIS). Genipin generates cross-

links spontaneously with protein, collagen, gelatin, chitosan, etc.

For a long time, genipin has been used as a treatment for choles-

tasis and hepatitis in traditional Chinese medicine. It has even

been used as a natural dye because of its tinting properties

with proteinic textiles [10]. Genipin itself has no color, but

forms blue particles by spontaneous reaction with amino acids

and protein. These characteristics of genipin have allowed it to

be used as a natural dye for textiles and for food. Many studies

have noted the remarkably low cytotoxicity and genotoxicity of

genipin compared to glutaraldehyde [11,12].


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Fig. 3. Touyama’s group’s presumed mechanism for the reaction of genipin with methylamine [9].

Sung et al. cultured human foreskin fibroblasts with geni-

pin, glutaraldehyde and epoxy compounds added and found

out that genipin has markedly low cytotoxicity as evaluated

by a difference in cell density [4]. Chang et al. also reported

that genipin has much less toxicity than glutaraldehyde and

the in vivo biocompatibility of heterograft tissue fixed with

genipin is far better [13,14]. Somers et al. cultured chicken

embryo fibroblasts in genipin and glutaraldehyde to estimate

their cytotoxicity. They found that a high concentration of

genipin has less toxicity than a low concentration of gluta-

raldehyde [15]. Huang et al. injected heterograft tissue fixed

with genipin into mice subcutaneously and cultured it. They

reported that heterograft tissue fixed with genipin caused less

inflammation and contained less calcium compared to tissue

fixed with glutaraldehyde [16].

It has not yet understood in detail and studies are in prog-

ress concerning the crosslinking mechanism between the gela-

tin in genipin and the molecule containing the primary amine.

Touyama et al. observed a reaction mechanism between

genipin and methylamine. Nucleophilic attack by primary

amine occurs at the C3 carbon atom in genipin and the sec-

ondary amine is attached to the aldehyde group [17]. Then

the collagen fiber is stabilized and the thermal stability is al-

so raised through intra/intermolecular crosslinking as above.

Fig. 3 shows a diagram of the reaction mechanism. As it not-

ed in Fig. 4, Chang et al. suggested that there was stable

bond among collagen fibers through intra/intermolecular

crosslink structure of tissue fixed with genipin and reaction

mechanism between genipin and amino acid group [18].

The aim of this study is to evaluate the possibility of using

genipin, a material for the fixation of heterograft tissue

(bovine pericardium) used in cardiac surgery, as a natural

Jae Suk Yoo, et al

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Fig. 4. (A) Presumable mechanism of amino group with genipin. (B) Schematic illustration of the presumable intramolecular and inter-molecular crosslinking structures of genipin-fixed tissue [18].

crosslink reagent biologically and biochemically modifiable

for clinical purposes.

Even on the assumption that genipin has less cytotoxicity,

we still need to confirm the physical, and mechanical character-

istics and thermal stability are at least equivalent to gluta-

raldehyde to be able to use genipin for fixation. Sung et al.

compared the denaturation temperature of three groups of por-

cine pericardial tissue, each fixed with glutaraldehyde, genipin,

or epoxy compound. They found that all three materials

showed a higher denaturation temperature compared to fresh

porcine pericardial tissue and all are effective crosslink agents

[4]. Although patches fixed with genipin showed a slightly


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lower temperature than patches fixed with glutaraldehyde in the

thermal stability test, there was no statistically significant

difference. There was also no significant correlation by concen-

tration of genipin, organic solvent, or timing of adding organic

solvent. In addition, the groups fixed with genipin showed an

equivalent level of tensile strength to the groups fixed with glu-

taraldehyde as a result of the tensile strength test.

Therefore, it can be concluded that genipin has potential as

an alternative crosslinking reagent to glutaraldehyde, pending

clinical studies.

CONCLUSION

By previous studies, genipin has been well known to be

less cytotoxic than glutaraldehyde. The present study showed

that bovine pericardial tissues fixed with genipin are com-

parable with those fixed with glutaraldehyde in mechanical

characteristics, tensile strength, and thermal stability. Thus,

we can consider genipin to be a possible alternative to gluta-

raldehyde as a crosslinking agent for fixing heterograft tissue.

However, the long-term physical and biochemical effects in

the human body and the immune response caused by peri-

cardial patches fixed with genipin are not yet known, so fur-

ther studies and animal experiments are required before

application.

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